Human cytomegalovirus multiple-strain infections and viral population diversity in haematopoietic stem cell transplant recipients analysed by high-throughput sequencing.

Human cytomegalovirus (HCMV) is an important opportunistic pathogen in allogeneic haematopoietic stem cell transplant (HSCT) recipients. High-throughput sequencing of target-enriched libraries was performed to characterise the diversity of HCMV strains present in this high-risk group. Forty-four HCMV-DNA-positive plasma specimens (median viral input load 321 IU per library) collected at defined time points from 23 HSCT recipients within 80 days of transplantation were sequenced. The genotype distribution for 12 hypervariable HCMV genes and the number of HCMV strains present (i.e. single- vs. multiple-strain infection) were determined for 29 samples from 16 recipients. Multiple-strain infection was observed in seven of these 16 recipients, and five of these seven recipients had the donor (D)/recipient (R) HCMV-serostatus combination D + R + . A very broad range of genotypes was detected, with an intrahost composition that was generally stable over time. Multiple-strain infection was not associated with particular virological or clinical features, such as altered levels or duration of antigenaemia, development of acute graft-versus-host disease or increased mortality. In conclusion, despite relatively low viral plasma loads, a high frequency of multiple-strain HCMV infection and a high strain complexity were demonstrated in systematically collected clinical samples from this cohort early after HSCT. However, robust evaluation of the pathogenic role of intrahost viral diversity and multiple-strain infection will require studies enrolling larger numbers of recipients.

Correction to: Advanced Robotic Therapy Integrated Centers (ARTIC): an international collaboration facilitating the application of rehabilitation technologies.

The original article [1] contains a small mistake concerning the ARTIC Team members mentioned in the Acknowledgements. The team member, Rocco Salvatore Calabrò had their name presented incorrectly. This has now been corrected in the original article.

Effectiveness of robot-assisted gait training in children with cerebral palsy: a bicenter, pragmatic, randomized, cross-over trial (PeLoGAIT).

BACKGROUND: Walking ability is a priority for many children with cerebral palsy (CP) and their parents when considering domains of importance regarding treatment interventions. Partial body-weight supported treadmill training has become an established therapeutic treatment approach to address this demand. Further, new robotic rehabilitation technologies have increasingly been implemented in the clinical setting to allow for longer training sessions with increased step repetitions while maintaining a consistent movement pattern. But the current evidence about its clinical effectiveness in pediatric rehabilitation is weak. The aim of this research project is therefore to investigate the effectiveness of robot-assisted gait training on improvements of functional gait parameters in children with cerebral palsy. METHODS/DESIGN: Children aged 6 to 18 years with bilateral spastic cerebral palsy who are able to walk at least 14 m with or without walking aids will be recruited in two pediatric therapy centers in Switzerland. Within a pragmatic cross-over design with randomized treatment sequences, they perform 5 weeks of robot-assisted gait training (three times per week with a maximum of 45 min walking time each) or a 5-week period of standard treatment, which is individually customized to the needs of the child and usually consists of 1-2 sessions of physiotherapy per week and additional hippotherapy, circuit training as well as occupational therapy as necessary. Both interventions take place in an outpatient setting. The percentage score of the dimension E of the Gross Motor Function Measure-88 (GMFM-88) as primary outcome as well as the dimension D of the GMFM-88, 6-minute and 10-meter walking tests as secondary outcomes are assessed before and at the end of each intervention period. Additionally, a 5-week follow-up assessment is scheduled for the children who are assigned to the standard treatment first. Treatment effects, period effects as well as follow-up effects are analyzed with paired analyses and independent test statistics are used to assess carry-over effects. DISCUSSION: Although robot-assisted gait training has become an established treatment option to address gait impairments, evidence for its effectiveness is vague. This pragmatic trial will provide important information on its effects under clinical outpatient conditions. TRIAL REGISTRATION: ClinicalTrials.gov: NCT00887848 . Registered 23 April 2009.

Monitoring of adenovirus (ADV)-specific T cells in a boy with ADV pneumonia and disseminated disease after lung transplantation.

Human adenovirus (ADV) infections are the cause of severe morbidity and mortality in transplant recipients. Cidofovir (CDV) is the current standard antiviral treatment. We report the case of a 3-year-old boy after lung transplantation with severe ADV sepsis, who was monitored for ADV-specific T cells during his disease and recovery. A strong increase in ADV-specific T cells was accompanied by resolution of ADV in blood and bronchoalveolar lavage fluid. Antiviral treatment with CDV was individually adapted according to anti-ADV immune responses, which provides a new method for tailoring antiviral treatment in lung transplant recipients.

Guillain-Barré syndrome associated with autochthonous infection by hepatitis E virus subgenotype 3c.

In this report, we present a case of a 50-year-old immunocompetent man with Guillain-Barré syndrome (GBS) associated with an autochthonous hepatitis E virus (HEV) infection. The patient presented with tetraparesis and elevated liver enzymes. HEV infection was confirmed serologically and by polymerase chain reaction (PCR) from blood and stool. Phylogenetic analysis revealed a novel HEV genotype 3 isolate closely related to other subgenotype 3c isolates from pig livers purchased in Germany. This indicates an autochthonous, potentially food-related hepatitis E and is, to our knowledge, the first report about a neurological syndrome associated with an HEV subgenotype 3c infection.

Evaluation of an automated real-time transcription-mediated amplification (TMA) assay for detection and quantification of cytomegalovirus DNA in different clinical specimens.

BACKGROUND: Reliable and fast detection and quantification of human cytomegalovirus (CMV) DNA in various diagnostic specimens is essential for care of immunocompromised or congenitally infected individuals. OBJECTIVES: To evaluate the analytical and clinical performance of the Panther Aptima® CMV (Hologic) quantitative real-time transcription mediated amplification (TMA) assay. STUDY DESIGN: Performance of the TMA assay run on the Hologic Panther Fusion was analysed for 32 proficiency testing samples and 21 quantitative reproducibility panel samples; additionally, we compared results of TMA assay and routine quantitative real-time PCR assays ("PCR-A"= Biomérieux CMV R-gene® or "PCR-B"= Laboratory-developed CMV-PCR) in 518 diagnostic specimens (254 plasma, 120 EDTA whole blood, 43 urine, 45 amniotic fluid and 56 breast milk) at two university hospital laboratories. RESULTS: All proficiency panel samples were correctly identified and quantified by the TMA assay; replicate testing of the reproducibility panel samples showed good reproducibility within and between the two laboratories. Sensitivity in plasma and WB was higher for the TMA assay detecting low-level CMV-DNAemia in samples tested negative by routine PCR. Quantitative CMV-DNAemia values correlated well between TMA and real-time PCR. Similarly, urine, AF and BM specimens showed a high rate of concordant results (91%, 98% and 98%, respectively) among TMA and PCR with good correlation of quantitative values. CONCLUSION: The performance of the Aptima® CMV TMA assay for viral blood load testing compared well to established real-time PCRs. In addition, it can be useful for diagnostics in urine, amniotic fluid and breast milk specimens.

Clinical significance of quantitative cytomegalovirus detection in bronchoalveolar lavage fluid in lung transplant recipients.

OBJECTIVES: Cytomegalovirus (CMV) is an important pathogen after lung transplant (LTx) and may be associated with bronchiolitis obliterans syndrome (BOS). We assessed the outcome of LTx patients with CMV DNA-positive bronchoalveolar lavage (BAL) during surveillance. METHODS: A single-center retrospective study was performed. Outpatients transplanted between September 2007 and February 2010, who had undergone at least 3 BALs, were included. CMV DNA load in BAL was measured by polymerase chain reaction (PCR). Monitoring of peripheral blood antigenemia was performed simultaneously. RESULTS: In total, 103 LTx patients underwent 1118 BALs. During median follow-up of 639 days (interquartile range, 495-780), 53 (51%) patients exhibited at least 1 positive CMV PCR in BAL. The incidence of positive CMV PCR varied post transplantation, with 6%, 30%, and 25% of BALs testing positive at <6 months, 6-24 months, and >24 months, respectively. Neither CMV BAL positivity, positive CMV antigenemia, nor dual positivity were significantly associated with BOS-free survival during follow-up. Patients with CMV-positive BAL more frequently developed CMV antigenemia in the first year (44% vs. 5%, respectively, log-rank P < 0.001). CONCLUSIONS: Detection of CMV-positive BAL after the sixth month appears common, but did not correlate with BOS-free survival after LTx in this study. An increased risk of developing blood antigenemia was observed in patients with positive CMV PCR in BAL, warranting close follow-up.

Enrichment of regulatory T cells in acutely rejected human liver allografts.

Acute cellular rejection (ACR) occurs frequently after liver transplantation and can usually be controlled. Triggering of allospecific immune responses and lack of immunoregulation are currently suggested as a cause of ACR, but there are no investigations of intrahepatic immune responses during ACR. Therefore we prospectively analyzed the intrahepatic T cell infiltration pattern in correlation to the severity of ACR in a cohort of patients with graft hepatitis (n = 151). While CD4(+) cells dominated the portal infiltrates in mild-moderate ACR, CD8(+) cells prevailed in severe ACR. Furthermore portal CD8(+) and not CD4(+) infiltration correlated with serum transaminases and with the likelihood of subsequent ACRs. Surprisingly, the rise of portal effector T cells density during ACR was surpassed by the increase in portal infiltration of regulatory T cells by a factor of two. Thus ACRs rather showed an increase and not a lack of regulation, as was suggested by analysis of peripheral blood mononuclear cells. Despite the pattern of enhanced immunoregulation, patients with severe ACR had a higher risk for subsequent rejections and showed a trend to a reduced survival. Thus, patients with severe rejections might need a modification of their immunosuppression to improve prognosis.

Chronic hepatitis e in heart transplant recipients.

Chronic courses of hepatitis E virus (HEV) infections have been described in immunosuppressed patients. We aimed to study the role of HEV infections in heart transplant recipients (HTR). 274 HTR were prospectively screened for HEV infection using an anti-HEV-IgG ELISA and HEV-PCR. In addition, 137 patients undergoing cardiac surgery (non-HTR) and 537 healthy subjects were studied cross-sectionally. The anti-HEV-IgG seroprevalence was 11% in HTR, 7% in non-HTR and 2% in healthy controls (HTR vs. healthy controls p<0.0001; non-HTR vs. healthy controls p<0.01). Anti-HEV tested positive in 4.0% in control cohorts of other immunocompromised patients (n = 474). Four HTR (1.5%) were chronically infected with HEV as shown by HEV-PCR and all four patients had liver transaminases of >200 IU/L and histological or clinical evidence of advanced liver disease. In three patients ribavirin treatment was successful with a sustained biochemical and virological response while treatment failed in one cirrhotic patient after ribavirin dose reduction. Heart transplant recipients and patients undergoing cardiac surgery have an increased risk for HEV infections. Chronic hepatitis E may explain elevated liver enzymes in heart transplant recipients. Treatment of HEV infection with ribavirin is effective but the optimal dose and duration of ribavirin therapy remains to be determined.

Improved quantitative PCR protocols for adenovirus and CMV with an internal inhibition control system and automated nucleic acid isolation.

With the establishment of routine virus load (DNAemia) screening for Human adenovirus (HAdV) and Cytomegalovirus (CMV) in post-transplant care quality standards for quantitative PCR-assays are increasing. Established real-time PCR assays were improved with a fully automated DNA-extraction and with a competitive internal control DNA packaged into a lambda phage which serves as an extraction and amplification control in each sample. HAdV and CMV DNA were detected and quantified simultaneously in various types of diagnostic samples like blood, feces or respiratory tract materials. Inhibition was observed in 0.33-0.66% of over 14,000 diagnostic samples, an infrequent but nevertheless not negligible event, which is observed mainly in stool samples. CMV viral load in broncho-alveolar lavage fluid (BALF) ranged between positive but below the quantitation limit of 1,000 copies/ml up to 1.8 × 10(7) copies/ml with a median of 6.0 × 10(3) copies/ml. Forty-one (4.7%) BALF samples had a viral load above 5.0 × 10(5) copies/ml, which was proposed as a threshold for the diagnosis of pneumonia. HAdV viral loads ranged between positive but below the quantitation limit of 1,000 copies/ml to a very high concentration of 1.3 × 10(11) copies/ml in stool and BALF samples. A HAdV-DNAemia of >10(4) copies/ml was found only in patients with stool viral load of above 10(5) copies/ml. These data support the hypothesis that quantitation in diagnostic materials other than blood may give valuable diagnostic information and that further evaluation of this approach is reasonable.

Chronic hepatitis E in hematopoietic stem cell transplant patients in a low-endemic country?

Cases of chronic hepatitis E have been described in patients after kidney and liver transplantation. In addition, hepatitis E virus (HEV) reactivation was reported after hematopoietic stem cell transplantation (HSCT). We here evaluated if HEV infection might explain elevated liver enzymes in a well selected cohort of allogeneic HSCT patients with biochemical evidence of hepatitis (n = 52). Of note, none of the subjects tested positive for HEV RNA, including 2 HSCT patients who had been infected with HEV already before transplantation. Thus, both chronic courses of HEV infections and HEV reactivations seem to be rather rare events in HSCT patients in a non-endemic country.

Hepatitis E: an emerging infectious disease in Germany?

Increased frequencies of HEV infections have been reported in several industrialized countries. We suggest that this finding might be explained by a better awareness of the disease and not by an increased incidence. Although reported HEV infections increased more than 6-fold in Germany in recent years, the seroprevalence remained unchanged (2 %).

[CMV-enterocolitis as a cause for repeated intestinal intussusceptions in an adult patient after liver transplantation?]. / CMV-Enterokolitis bei einer erwachsenen lebertransplantierten Patientin als Ursache rezidivierender Invaginationen?

Intestinal intussusception in the adult is often idiopathic but also known to be associated with chronic inflammatory bowel disease, coeliac disease, tumours or previous abdominal operations. A 22-year-old women after liver transplantation due to Crigler Najar Syndrome suffered from repeated episodes of abdominal pain. The diagnosis of repeated self-limited intestinal intussusceptions was made by computed tomography and ultrasonography. A laparoscopy revealed no cause for the intussusceptions. During a new episode of abdominal pain caused again by an intussusception a colonoscopy was performed that showed aspects of a discreet colitis. In the biopsies CMV was detected by qualitative PCR, while blood tests for CMV pp65 antigen were negative. A therapy with gancyclovir was initiated which lead to remission of the patient's symptoms. A colonoscopy six weeks later showed a completely normal colon, while in the biopsies CMV was not detectable. After a follow-up of one year the patient has not suffered from any further episodes. This case demonstrates the role of chronic intestinal CMV infection as a possible causative factor for repeated intussusceptions in immunosuppressed patients. Whenever possible a PCR for CMV in colon biopsies should be carried out to detect an intestinal CMV infection because as shown in our case results for immunohistopathology and CMV pp65 can be negative despite a chronic infection.

[Adenovirus infections]. / Adenovirusinfektionen: Buntes Bild von Krankheiten durch eine Vielzahl von Virustypen.

During childhood several adenovirus infections have to be experienced, each with another of the multiple types. The spectrum of associated diseases ranges from endemic, mild and self-limiting upper respiratory tract infections to epidemic gastroenteritis, eye infections, cystitis and less frequently atypical pneumonia and severe infections of internal organs; however, the vast majority of adenovirus infections during childhood are not life threatening with the exception of the infrequent infections of neonates and the frequent infection of hematopoietic stem cell recipients (disseminated disease).

Frequency, Characteristics and Risk Factors of Aggressive Incidents in a Paediatric Rehabilitation Setting: A Prospective Survey.

Aim: Aggressive incidents (AI) are a serious concern in health care and can have negative effects on the physical and emotional well-being staff. This study aimed to determine frequency, characteristics and risk factors for aggressive behavior.Methods: AI were recorded during six months by the staff in a pediatric rehabilitation clinic using the evaluation form for AI (EVA). Patients were divided into the study group (patients who were involved in AI) and controls.Results: 14/105 (13%) of patients were involved in 79 AI. 0.44 AI per day occurred. Most often AI occurred on Mondays and 98% included physical, 22% verbal aggression. Most frequent target (43%) were nurses, followed by therapists (31.6%).Significant risk factors for AI were: previous aggressive behavior (p = .038), lower cognitive and higher mobility sub-scores in the WeeFIM. Conclusion: Findings emphasize the magnitude of AI in pediatric rehabilitation and thus the importance of implement preventive strategies.Abbreviations: ADL: Activity of daily living; AI: Aggressive Incidents; CFCS: Communication Function Classification System; EVA: Recording aggressive incidents (Erfassung von Aggressionsereignissen); GMFCS: Gross Motor Function Classification System; MACS: Manual Ability Classification System; SOAS-R: Staff Observation of Aggression Scale-Revised; WeeFIM: Functional Independence Measure for Children; WPV: Workplace violence.

Severe underquantification of HIV-1 group O isolates by major commercial PCR-based assays.

HIV-1 diversity poses major challenges to viral load assays because genetic polymorphisms can impede nucleic acid detection. In addition to the on-going viral diversification within the HIV-1 group M pandemic, HIV-1 genetic diversity is further increased by non-group M infections, such as HIV-1 groups O (HIV-1-O), N and P. We here conducted a systematic evaluation of commercially available PCR assays to detect HIV-1-O isolates. We collected 25 primary HIV-1-O isolates covering all genetic clusters within HIV-1-O. Subsequently, this panel of isolates was tested on eight commercially available quantitative and five qualitative HIV-1 PCR-based assays in serial dilutions. Sequence analyses were performed for severe cases of underquantification or lack of detection. We observed differences between the assays in quantification that depended on the HIV-1-O isolate's subgroup. All three tested HIV-1-O subgroup IV isolates were underquantified by the Roche CAP/CTM >800-fold compared to the Abbott RealTime assay. In contrast, the latter assay underquantified several subgroup I isolates >200-fold. Notably, the Xpert HIV-1 Viral Load test from Cepheid failed to detect two of the HIV-1-O isolates, whereas the Roche Cobas 8800 assay readily detected all isolates. Comparative sequence analyses identified polymorphisms in the HIV-1-O long-terminal repeat and integrase genes that likely underlie inadequate nucleic acid amplification. Potential viral load underquantification should be considered in therapeutic monitoring of HIV-1-O-infected patients. Pre-clinical assessments of HIV-1 diagnostic assays could be harmonized by establishing improved and internationally standardized panels of HIV-1 isolates that cover the dynamic diversity of circulating HIV-1 strains.

New real-time PCR detects prolonged norovirus excretion in highly immunosuppressed patients and children.

Noroviruses (NoV) are a major cause of epidemic nonbacterial gastroenteritis and affect all age groups worldwide. Three of five NoV genogroups, namely, genogroup I (GI), GII, and GIV, are associated with human disease. Unfortunately, these genogroups demonstrate a high degree of sequence diversity, complicating the design of pan-NoV diagnostic PCR tests. To decrease the risk of false-negative test results, we have developed a new one-step real-time TaqMan reverse transcription-PCR protocol. This protocol detects all human NoV genogroups in one reaction with a sensitivity of 400 virus genome equivalents/reaction for both GI and GII. The use of in vitro-transcribed NoV RNA as an external standard allows (semi)quantification of viral loads in samples. In a retrospective analysis of 206 stool samples from 77 patient episodes, the duration of NoV excretion and the amount of virus excreted were determined. Twenty (26.0%) of these episodes lasted longer than 10 days. Univariate risk factor analysis revealed the patient status after organ transplantation (odds ratio [OR], 7.49 [95% confidence interval, 2.06 to 28.32]; P < 0.001), immunosuppression (OR, 9.19 [95% confidence interval, 2.50 to 35.39]; P < 0.001), and age of less than 10 years (OR, 4.58 [95% confidence interval, 1.36 to 15.77]; P = 0.004) as risk factors for a NoV excretion period of more than 10 days. These findings were confirmed by time-dependent Kaplan-Meier analyses, whereas multivariate Cox regression analyses identified immunosuppression as the sole risk factor. Surprisingly, in contrast to the excretion periods, the viral loads in stools did not increase in connection with age or immunosuppressive status. This fact may be one important piece in the pattern of high-level NoV transmissibility and may have an impact on the development of transmission prevention strategies.

[ADVANCE: a morbidity mortality study of diabetes and hypertension]. / Hypertension et diabète. ADVANCE: une étude de morbidité-mortalité.

The ADVANCE study is a morbidity-mortality double-blind trial carried out in normotensive or hypertensive patients with type 2 diabetes. The patients were randomly assigned to receive containing a fixed-combination tablet of an ACE inhibitor (perindopril) with a diuretic (indapamide) (4 mg/l,250 mg, n=5569), or placebo (n=5571), administered if needed on top of other blood pressure lowering agents. Significant reductions in the relative risk of death from cardiovascular disease (18%), total coronary events (14%), and total renal events (21%) were observed. Thus, in patients with type 2 diabetes, a drug regimen based on a fixed-dose combination of perindopril/ indapamide affords major protection against both the macro and microvascular complications.

Risk groups for clinical complications of norovirus infections: an outbreak investigation.

Norovirus infections have been described as self-limiting diseases of short duration. An investigation of a norovirus outbreak in a university hospital provided evidence for severe clinical features in patients with several underlying diseases. Clinical outcomes of norovirus infection were defined. Risk-factor analysis targeting underlying diseases and medication was performed using multivariate analyses. In five outbreak wards, 84 patients and 60 nurses were infected (an overall attack rate of 32% in patients, and 76% in nurses). The causative agent was the new variant Grimsby virus. Severe clinical features, including acute renal failure, arrhythmia and signs of acute graft organ rejection in renal transplant patients, were observed in seven (8.3%) patients. In multivariate analyses, cardiovascular disease (OR 17.1, 95% CI 2.17-403) and renal transplant (OR 13.0, 95% CI 1.63-281) were risk-factors for a potassium decrease of >20%. Age >65 years (OR 11.6, 95% CI 1.89-224) was a risk-factor for diarrhoea lasting >2 days. Immunosuppression (OR 5.7, 95% CI 1.78-20.1) was a risk-factor for a creatinine increase of >10%. Norovirus infections in patients with underlying conditions such as cardiovascular disease, renal transplant and immunosuppressive therapy may lead to severe consequences typified by decreased potassium levels, increased levels of C-reactive protein and creatine phosphokinase. In the elderly, norovirus infection may lead to an increased duration of diarrhoea. Therefore patients at risk should be hospitalised early and monitored frequently. Strict preventional measures should be implemented as early as possible to minimise the risk of nosocomial outbreaks.

Transient induction of cytokine production in human myocardial fibroblasts by coxsackievirus B3.

Cytokine expression in enterovirus infections of the heart may trigger inflammation and have detrimental effects on myocytes. However, the induction of cytokines in human myocardial cells by cardiotropic enteroviruses, for example, Coxsackievirus B3 (CVB3), was not yet demonstrated. Fibroblasts are the predominant cell type of the myocardial interstitium before inflammatory infiltration develops. Hence, we investigated, by enzyme immunoassays, reverse transcription-quantitative polymerase chain reaction (RT-qPCR), and nucleic acid sequence-based amplification (NASBA), whether CVB3 induces cytokine expression in cultured human myocardial fibroblasts. As early as 3 hours after infection, RT-qPCR demonstrated a 2-fold increase of interleukin (IL)-6 and IL-8 mRNA compared with basal transcription, resulting in a significant increase of IL-6 and IL-8 to a median level of 1500 pg/mL (range, 1246 to 1858) and 529 pg/mL (range, 428 to 601) in culture supernatants, respectively. IL-6 and IL-8 expression returned to basal levels within 3 and 5 days, respectively, despite a persistent (carrier-state) CVB3 infection. For comparison, IL-6 and IL-8 were induced in dermal fibroblasts later than 3 days after CVB3 infection. Although the low-level IL-1alpha transcription of myocardial fibroblasts was not significantly increased, IL-1alpha was released from cells to culture supernatants 5 days after infection. Furthermore, a suppression of interferon-beta transcription was demonstrated up to 24 hours after CVB3 infection of myocardial fibroblasts by highly sensitive NASBA. In conclusion, our results demonstrate a heart-specific pattern of a rapid and transient induction of proinflammatory cytokines after CVB3 infection, whereas the expression of protective interferon-beta was suppressed by CVB3.